FIGURE 4.

Exogenous cathepsin (Cath) L enhances the entry of SARS/MLV, but not that of NL63/MLV.A, HEK293T cells were transfected with cathepsin B, L, or S expressor plasmids and various amounts of human ACE2 plasmid as described under”Experimental Procedures.“Cells were divided and replated for infection, metabolic labeling, or flow cytometry the following day. Two days post-transfection cells were incubated with SARS/MLV, NL63/MLV, or VSV-G/MLV. In parallel, cell surface ACE2 expression was assessed by flow cytometry with anti-human-ACE2 antibody. Infection by GFP-expressing pseudovirions, measured 2 days post-infection by flow cytometry (vertical axis), is plotted here against ACE2 expression (horizontal axis). B, approximately 36 h post-transfection, an aliquot of transfected cells used in A was labeled overnight with [³⁵S]cysteine and [³⁵S]methionine. Cells were lysed in 0.5% Nonidet P-40/PBS containing protease inhibitor mixture, immunoprecipitated with 1D4 antibody recognizing a C-terminal epitope present on the cathepsin protein, and analyzed by SDS-PAGE. Three lanes each for cathepsins B, L, and S represent three transfections (in the order of increasing amounts of ACE2 DNA). C, MEFs cultured from a cathepsin L^–/– mouse were transduced with VSV-G/MLV carrying the human ACE2 gene and, 5 h later, with VSV-G/MLV carrying the human cathepsin L gene. Cells were split and infected with SARS/MLV, NL63/MLV, MARV/MLV, Ebola/MLV, or VSV-G/MLV. Infection efficiency was assessed 3 days later by measuring GFP expression by flow cytometry and is expressed as a percentage of GFP-expression observed with cells transfected with ACE2 alone.